Abstract

Stressful situations provoke the fight-or-flight response, incurring rapid elevation of cardiac output via activation of protein kinase A (PKA). In this issue of the JCI, Yang et al. focus on the L-type calcium channel complex (LTCC), and their findings require reexamination of dogmatic principles. LTCC phosphorylation sites identified and studied to date are dispensable for PKA modulation of LTCC; however, a CaVβ2-CaV1.2 calcium channel interaction is now shown to be required. Yang et al. suggest a new hypothesis that LTCC modulation involves rearrangement of auxiliary proteins within the LTCC. However, we still do not know the targets of PKA that mediate LTCC modulation.

Authors

Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant α1C subunits lacking capacity to bind CaVβ can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenergic pathway agonists, and thus adrenergic augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a β-subunit–sequestering peptide sharply curtailed β-adrenergic stimulation of WT Ca2+ channels, identifying an approach to specifically modulate β-adrenergic regulation of cardiac contractility. Our data demonstrate that β subunits are required for β-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.

Reconstitution experiments concluded that binding to β subunits is indispensable for α1C trafficking to the cell surface (6–14). The physiological relevance of this finding was initially supported by β2 knockout mice, which were embryonic lethal, likely secondary to a decreased L-type Ca2+ current (15). An initial idea that β binding to the α-interaction domain (AID) of the α1-subunit I-II loop shielded an ER retention signal in the I-II loop to allow forward trafficking of the channel proved inadequate in subsequent experiments (9, 16–18). Surprisingly, cardiomyocyte-specific, conditional deletion of the Cacnb2 gene in adult mice reduced β2 protein by 96% but caused only a modest 29% reduction in Ca2+ current, with no obvious cardiac impairment (19). Interpretation of this result is ambiguous, however, as it is complicated by the remnant (~4%) β2 expression as well as the presence other CaVβ isoforms expressed in adult cardiomyocytes (13). Moreover, a contrasting viewpoint was provided by a study in which shRNA-mediated knockdown of β2 in adult rat myocytes substantially diminished Ca2+ current (20).

To definitively address the controversies regarding the role of β subunits in mediating trafficking and regulation of Ca2+ channels in the heart, we created transgenic mice lines with 3 mutations in the AID, which renders the pore-forming α1C subunit incapable of binding β subunits. With this new model, we definitively demonstrate in vivo that β subunit binding to α1C is not required for trafficking and that the basal function of β-less Ca2+ channels is only minimally altered.

Instead, we found that the β subunit is obligatory for transducing β-adrenergic signals to cardiac CaV1.2 channels. Cardiac CaV1.2 channels are prominently upregulated by β-adrenergic agonists via activation of protein kinase A (PKA) (21, 22) as part of the fundamental flight-or-fight response, yet the detailed mechanisms by which PKA activates CaV1.2 remain unknown despite several decades of investigation. Recently, we reported that alanine substitution of all consensus, conserved PKA phosphorylation sites (> 22 serines/ threonines) in the α1C subunit did not affect adrenergic regulation of CaV1.2 in vivo (23). Prior studies also ruled out a contribution for the β subunit, as substitution or elimination of potential PKA phosphorylation sites did not perturb β-adrenergic regulation (24–27), although other consensus PKA sites are present in the N-terminal regions of the protein. We found that β subunit binding to α1C, but not PKA phosphorylation of β, is absolutely essential for the augmentation of Ca2+ current and cardiac contractile response to β-adrenergic PKA stimulation. These findings identify the key regulatory mechanisms impacting β-adrenergic regulation of Ca2+ influx and contractility in the heart.

We assessed the impact of loss of β binding on AID-mutant α1C subcellular localization and functional expression in cardiomyocytes using 3 complementary approaches. First, immunofluorescence experiments using anti–FLAG antibody on fixed cardiomyocytes indicated that both transgenic pWT α1C and AID-mutant α1C channels displayed a similar striated pattern consistent with surface membrane distribution and localization in transverse tubules (Figure 1D). Second, we exploited the T1066Y/Q1070M mutations that impart relative DHP-resistance (32, 33) to block Ca2+ currents from endogenous DHP-sensitive CaV1.2 with nisoldipine and isolate Ca2+ current from transgenic pWT α1C or AID-mutant α1C channels. Compared with cardiomyoctes isolated from NTG control mice, cardiomyocytes isolated from both pWT and AID-mutant α1C transgenic mice had increased peak Ca2+ currents, and substantial peak Ca2+ currents remaining after exposure to nisoldipine (Figure 1, E and F). Third, field-stimulated contraction of cardiomyocytes isolated from transgenic AID-mutant α1C mice persisted in the presence of 300 nM nisoldipine (Figure 1, I and J), similar to the contraction of cardiomyocytes isolated from transgenic pWT α1C mice in the presence of nisoldipine. Contraction of cardiomyoyctes isolated from NTG was markedly inhibited by nisoldipine (Figure 1, G and H). Overall, these results demonstrate that transgenic β-less AID-mutant α1C channels traffic to the sarcolemma and trigger E-C coupling in cardiomyocytes. This is in stark contrast to the necessary role of β binding for surface trafficking and function of CaV1.2 channels reconstituted in heterologous cells (Supplemental Figure 1, A and B), or expressed in hippocampal neurons (34).

We also considered that endogenous WT α1C channels could couple with AID-mutant α1C channels to facilitate trafficking of β-less channels to the surface membranes in cardiomyocytes, which could be the basis for the observed differences between cardiomyocytes and heterologous expression systems. To determine whether coupling-induced trafficking could occur, we coexpressed either DHP-resistant pWT α1C or DHP-resistant AID-mutant α1C with both WT α1C and β2 subunits in tsA201. In the presence of nisoldipine, which inhibits the WT α1C channels, tsA201 cells expressing the AID-mutant α1C channels had no remaining Ca2+ current (Supplemental Figure 1C, right), whereas cells expressing the DHP-resistant pWT α1C had remaining current (Supplemental Figure 1C, left), implying that at least in tsA201 cells, β-less channels were unable to “hitchhike” to the membrane with WT channels.

PKA modulation of CaV1.2 channels is dependent on α1C-β interactions. In heterologous expression studies, β subunits not only enable α1C surface trafficking, but also can differentially induce, depending on β subunit isoform, a hyperpolarizing shift in the voltage dependence of CaV1.2 activation and increase the channel open probability (Po) (12, 27). We assessed the biophysical properties of the transgenic β-less AID-mutant α1C channels compared with transgenic pWT Ca2+ channels. Surprisingly, normalized current-voltage (I-V) relationships of nisoldipine-resistant transgenic pWT and AID-mutant α1C channels were remarkably similar (Figure 2A). The midpoint potentials, derived from a Boltzmann function, for steady-state activation demonstrated a small, nonsignificant 1-mV hyperpolarizing shift for the AID-mutant channels compared with control pWT channels (Figure 2B), whereas the slope factors for the 2 channel types were not different (Figure 2C). Furthermore, the inactivation kinetics of nisoldipine-resistant Ca2+ currents were not significantly different at any test potential between cardiomyocytes isolated from pWT and AID-mutant α1C, respectively (Figure 2D). Therefore, in adult cardiomyocytes, CaV1.2 channels comprised of transgenic β-less α1C have similar voltage dependence of activation and inactivation kinetics as transgenic pWT CaV1.2 channels.

We next determined the sensitivity of CaV1.2 channels containing either transgenic pWT α1C or AID-mutant α1C to PKA modulation. In cardiomyocytes isolated from mice expressing transgenic pWT α1C, 200 nM isoproterenol increased the nisoldipine-insensitive current by a mean of 1.9-fold ± 0.1-fold (Figure 2, E–J), and shifted the Vmid in the hyperpolarizing direction by a mean of 4.4 mV (Figure 2B). Similarly, forskolin, which directly activates adenylyl cyclase, thereby bypassing β-adrenergic receptors, increased transgenic pWT α1C Ca2+ currents by 1.8-fold ± 0.1-fold (Figure 2, H–J). In sharp contrast, Ca2+ currents through transgenic AID-mutant α1C CaV1.2 channels were insensitive to either isoproterenol (Figure 2, B, F, G, I, and J) or forskolin (Figure 2, H–J). In cardiomyocytes, there is an inverse relationship between total peak current and isoproterenol-induced or forskolin-induced fold increase in Ca2+ current (27). In cardiomyocytes isolated from transgenic pWT α1C mice, we observed an inverse relationship between basal current density and isoproterenol- or forskolin-induced increase in Ca2+ current (Figure 2J). For the transgenic AID-mutant β-less channels, however, activation of PKA by either forskolin or isoproterenol had no effect on Ca2+ current, regardless of basal Ca2+ current density (Figure 2J).

To address whether the YWI/AAA mutations themselves produced an intrinsic insensitivity of the channel to PKA modulation, we sought to engender conditions under which there would be a predominance of β-less endogenous CaV1.2 channels in isolated cardiomyocytes. We achieved this by using adenovirus to overexpress a YFP-tagged 18-residue AID peptide derived from α1C I-II loop (or a mutant YWI/AAA peptide as a control) in cultured adult guinea pig ventricular cardiomyocytes. We reasoned that this intervention would serve as a sponge for endogenous β subunits, leaving a majority of endogenous CaV1.2 channels devoid of β. In control cells expressing either GFP or YFP-tagged mutant (YWI/AAA) AID peptide incapable of binding β, 1 μM forskolin resulted in a robust 4- to 5-fold increase in whole-cell current amplitude (Figure 3, A, C, D, and F–H). By contrast, this response was sharply curtailed in cardiomyocytes overexpressing YFP-AID peptide (Figure 3, B, E, G, and H). Hence, β-less WT α1C channels also demonstrate a marked insensitivity to PKA modulation.

We also considered 2 trivial explanations that could potentially account for the insensitivity of AID-mutant α1C to PKA stimulation: (a) these channels were already phosphorylated by PKA under basal conditions, or (b) the β-adrenergic signaling pathway was compromised in cardiomyocytes from AID-mutant α1C transgenic mice. To address whether transgenic AID-mutant α1C channels were basally PKA phosphorylated, we used a cell-permeable cAMP-PKA inhibitor (Rp-8-Br-cAMPS), which functions by occupying cAMP binding sites thereby preventing activation of PKA holoenzyme. Rp-8-Br-cAMPS reverses isoproterenol-mediated upregulation of endogenous CaV1.2 by approximately 96% (23). In transgenic AID-mutant mice cardiomyocytes, Rp-8-Br-cAMPS did not inhibit nisoldipine-resistant basal current (Supplemental Figure 2A), ruling out the idea that AID-mutant α1C channels were basally PKA phosphorylated. The integrity of the β-adrenergic pathway in transgenic AID-mutant mice cardiomyocytes was assessed by probing whether isoproterenol application led to phosphorylation of phospholamban, a well-known PKA target in the heart (35). Western blotting indicated that phospholamban was appropriately phosphorylated at Ser16 in response to isoproterenol (Supplemental Figure 2B), confirming that the β-adrenergic signaling pathway was intact in AID-mutant transgenic mice cardiomyocytes.

β-adrenergic regulation of CaV1.2 does not require PKA phosphorylation of β subunits. The simplest explanation for the necessary role of α1C-β interaction in PKA modulation of CaV1.2 is that the β-subunit contains phosphorylation site(s) that are vital to this regulation. Indeed, 2 phosphorylation sites on β2 C-terminus (Ser512 and Ser570) were previously identified and proposed to play a role in PKA modulation of CaV1.2 (36). However, a knockin mouse expressing a β2 subunit truncated after Pro501 displayed normal PKA modulation of CaV1.2, thus ruling out involvement of any putative C-terminal phosphorylation sites (24). Nevertheless, it remained possible that previously unappreciated phosphorylation sites N-terminal to Pro501 could mediate the increased CaV1.2 channel activity in response to activated PKA. Using both manual sequence analyses and several web-based PKA phosphorylation prediction tools (37–41), we identified 18 conserved consensus PKA phosphorylation sites in the N-terminus, SH3, and GK domains of human β2b (residues labeled red in Supplemental Figure 3). We mutated all 18 Ser/Thr residues to Ala in human β2b, and generated transgenic mice with inducible cardiomyocyte-specific expression of either GFP-tagged WT or 18-mutant β2b subunits using the same bitransgenic system as in Figure 1B. The WT and mutant β2b transgenic mice were fed doxycycline for 1 week, thus ensuring high levels of expression of the GFP-tagged β2 subunits (Figure 4A). We exploited the larger size of GFP-tagged β2 subunits compared with endogenous β to determine relative expression of transgenic and native β2 subunits (Figure 4B). Western blot indicated that in cardiomyocytes from transgenic mice, both GFP β2 and GFP-mutant β2 were markedly overexpressed (~9:1) compared with endogenous β2 (Figure 4C). Isoproterenol increased peak CaV1.2 current by a mean of 1.5-fold ± 0.1-fold in GFP-WT β2-expressing cells and 1.6-fold ± 0.1-fold in GFP-mutant β2-expressing cells, respectively, similar to nontransgenic mice (Figure 4, D–G). For both GFP-WT and GFP-mutant β2b Ca2+ channels, isoproterenol shifted the Vmid of steady-state activation by –7.0 mV and –7.5 mV, respectively. These data indicate that, although the α1C-β2 interaction is necessary for β-adrenergic regulation of CaV1.2, direct PKA phosphorylation of β2 is not involved.

β-adrenergic regulation of cardiac contractility requires PKA regulation of CaV1.2. We next exploited the findings that transgenic β-less AID-mutant α1C channels are insensitive to PKA modulation to probe the specific role of CaV1.2 modulation in the positive inotropic effect of β-adrenergic agonists in both isolated cardiomyocytes and in the whole heart. In transgenic pWT α1C cardiomyocytes, with endogenous CaV1.2 channels silenced with nisoldipine, isoproterenol produced a robust 100% increase in fractional shortening (Figure 5, A and C). By contrast, this response was severely diminished in cardiomyocytes expressing transgenic β-less AID-mutant α1C channels in which isoproterenol produced a relatively meager 25% increase in fractional shortening (Figure 5, B and C). Consistent with the effects of isoproterenol on phospholamban phosphorylation (Supplemental Figure 2B), isoproterenol enhanced relaxation in cardiomyocytes isolated from both pWT and AID-mutant α1C transgenic mice (Figure 5D).

We then assessed the role of CaV1.2 modulation in β-adrenergic agonist-induced positive inotropy at the whole-organ level by inserting a pressure-transduced balloon into the left ventricle of Langendorff-perfused transgenic mice hearts. This approach enabled measurement of cardiac contractility independent of vascular or systemic effects. Hearts were paced at 400 beats per minute to remove the potentially confounding effect of heart rate variability on contractility (42). After baseline measurements, 300 nM nisoldipine was infused into the coronary arteries via the aorta to suppress endogenous CaV1.2 channel currents. In hearts from nontransgenic mice, nisoldipine markedly reduced basal cardiac contractility due to the block of endogenous CaV1.2 channels (Figure 5E). In pWT α1C hearts, infusion of nisoldipine yielded a comparatively weaker effect on basal contractility owing to the expression of DHP-resistant Ca2+ channels (Figure 5F); a further infusion of 200 nM isoproterenol strongly increased cardiac contractility by 3.3-fold (Figure 5, G and I). By contrast, using the same experimental paradigm in hearts from β-less AID-mutant transgenic mice, the response to isoproterenol was nearly abolished, yielding an average increase in cardiac contractility of only 1.2-fold (Figure 5, H and I).

Much of our current understanding regarding mechanisms underlying CaV1.2 trafficking and modulation derives from studies on recombinant channels reconstituted in heterologous cells. These cells lack the complex cytoarchitecture and intracellular milieu of adult cardiomyocytes. Recently, we developed an approach that utilizes transgenic mice expressing doxycycline-inducible, cardiac-specific, DHP-resistant α1C. Compared with knockin mice models (43, 44), this approach is both cost-effective and rapid, and perhaps more importantly, enables us to induce brief expression of mutant channels in adults, permitting the comparison of WT and mutant α1C structure-function mechanisms in the absence of developmental abnormalities and heart failure. The titration of the level of CaV1.2 expression is important, as the magnitude of β-adrenergic stimulation of CaV1.2 is reduced with increased basal current density (27, 45–49). Stratifying the magnitude of β-adrenergic–mediated upregulation of CaV1.2 current by total basal current density attenuates this confounding variable (Figure 2J and Figure 4G).

Overall, we show that in cardiomyocytes, the AID motif is required for the high-affinity interaction between α1C and β subunits, and that β-less CaV1.2 channels traffic to the dyad and produce currents that mediate normal E-C coupling. The AID-mutant β-less Ca2+ currents were completely refractory to PKA activation. These findings, combined with our recent studies (23), fundamentally recast our views on mechanisms underlying CaV1.2 trafficking and PKA modulation in cardiomyocytes as they show that (a) it is possible for β-less channels to traffic to the cell surface, (b) β2 binding to α1C is indispensable for PKA modulation of CaV1.2, and that β-adrenergic regulation of CaV1.2 can be specifically attenuated by sequestering β subunits, and (c) conserved consensus PKA phosphorylation sites in α1C (23) and β2b are not required for β-adrenergic regulation of CaV1.2 in the heart. Further, we directly show that β-adrenergic modulation of CaV1.2 is critical for sympathetic augmentation of cardiac inotropy, which is essential for the fight-or-flight response.

When coexpressed with α1 subunits in heterologous expression systems such as Xenopus oocytes or human embryonic kidney (HEK) cells, β subunits markedly augment current density by increasing membrane targeting and altering electrophysiological properties (6–8). In the adult heart, however, Ca2+ channels can traffic to the surface membrane without binding to β. How β-less α1C channels traffic to the dyad in cardiomyocytes but not in a less complex system such as HEK cells is not yet clear. Although low-affinity interactions between heterologously expressed β subunit GK and SH3 domains and the CaV2.1 α subunit in oocytes have been described (50), these potential interactions do not appear to be sufficient to rescue the trafficking of AID-mutant CaV1.2 channels in tsA-201 cells. Moreover, conditional knockout of Cacnb2 in adult cardiomyocytes caused only a 29% reduction in current density (19).

Regardless of the mechanisms enabling trafficking to the cell surface, β-less CaV1.2 channels are functionally normal under basal conditions in adult cardiomyocytes. However, the β-less channels cannot be regulated by adrenergic-PKA stimulation, although the β subunit does not appear to be the functional target of PKA. To differentiate between the lack of β binding as opposed to the mutations in the AID as causative of the defect in β-adrenergic regulation of CaV1.2, we used the complementary approach of expressing using adenovirus, YFP-AID– and YFP-mutant AID–containing peptides in cultured adult guinea pig ventricular myocytes. The response to forskolin was markedly reduced by preventing β subunits from interacting with endogenous WT α1C, implying that lack of β binding to α1C is sufficient to prevent β-adrenergic regulation of CaV1.2 in the heart. Our studies cannot address where and when β subunits first interact with α1C subunits in the heart.

Identifying the functional PKA target is more complicated. It is likely not solely α1C, based on our prior studies eliminating all conserved consensus PKA phosphorylation sites in the α1C subunit (23). Likewise, it is not solely β, based on eliminating all conserved PKA phosphorylation sites in β2 (Figure 4). Thus, our findings suggest that either there is redundancy between α1C and β subunits, such that PKA phosphorylation of either subunit is sufficient to mediate adrenergic regulation of Ca2+ channels in the heart, or that PKA phosphorylation of the core CaV1.2 subunits, α1C and β, are not necessary for β-adrenergic regulation of the Ca2+ influx in the heart. This can be addressed by cross-breeding the transgenic mice harboring Ala substitutions of all PKA consensus sites in α1C and β2b. Although PKA phosphorylation of β is not required, β subunits, via binding to the I-II loop, could regulate pore opening and voltage-sensor movement. The domain I S6-AID linker forms a continuous helix that may act as a rigid rod through which β subunits modulate channel gating (51).

The loss of β-adrenergic activation of CaV1.2 correlated with a markedly attenuated β-adrenergic contractile response. Originally proposed by Fabiato, CaV1.2 current has 2 distinct roles in E-C coupling: triggering the release of Ca2+ from the sarcoplasmic reticulum (SR) and loading the cell (and SR) with Ca2+ (52). The loss of adrenergic regulation of CaV1.2 could affect both triggering of RyR2 and the loading of SR with Ca2+, thereby attenuating the adrenergically driven inotropic response. We believe that our findings are the first to demonstrate experimentally the vital role of β-adrenergic stimulation of CaV1.2 in shaping the flight-or-fight response in the heart, and validate a recently proposed mathematical model predicting that the loss of β-adrenergic stimulation of CaV1.2 would markedly limit Ca2+ transients and contraction (53). PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2 also enhances the open probability of the RyR2 Ca2+ release channels in the SR by enhancing their sensitivity to cytosolic (54) and synchronizing SR Ca2+ release (55–57). It remains controversial, however, as to whether increasing the open probability of RyR2 is critically important for inotropic responses in the heart (58–60). We demonstrate that without augmented CaV1.2 current to load the cell with additional Ca2+ and/or enhance RyR opening via Ca2+-induced Ca2+ release, β-adrenergic agonist-induced phosphorylation of RyR2 and phospholamban does not result in substantial β-adrenergic augmentation of cardiac contractility.

In summary, we have found that Ca2+ channel β-subunit binding to the pore-forming α1C subunit is not required for trafficking and function of the Ca2+ channel in the heart. The loss of α1C-β2 binding causes marked attenuation of β-adrenergic–induced stimulation of CaV1.2 and inotropy. Thus, we identify a new function for β subunits in the heart: as an essential component of the PKA-mediated augmentation of CaV1.2 and increased cardiac contractility that occurs during the physiological fight-or-flight response.

Reagents. Nisoldipine and Rp-8-Br-cAMPS were purchased from Santa Cruz Biotechnology. All other chemicals were acquired from MilliporeSigma.

Animals. The α1C transgenic constructs were generated by fusing rabbit Cacna1c cDNA (accession X15539) to the modified murine α-myosin heavy chain (MHC) tetracycline-inducible promoter (“responder” line) vector (gift of Jeffrey Robbins and Jeffrey Molkentin, University of Cincinnati, Cincinnati, OH) (61, 62). The α1C subunit was engineered to be both DHP insensitive with the substitutions T1066Y and Q1070M (32, 33) and tagged with a 3X-FLAG epitope. We made alanine substitutions of 3 conserved residues, Y467, W470, and I471, in the AID domain of rabbit α1C (Figure 1A). Two distinct AID-mutant α1C were created and studied. The results obtained from each of these lines were equivalent and therefore the data were pooled. The β2b transgenic constructs were generated by ligating a N-terminal GFP-tagged human CACNB2b cDNA (accession AAG01473) to the tetracycline-inducible vector. These mice were bred with cardiac-specific (αMHC), doxycycline-regulated, codon-optimized reverse transcriptional transactivator (rtTA) mice (obtained via the Mutant Mouse Resource and Research Center [MMRRC]) (63) to generate double-transgenic mice. The α1C transgenic animals received 0.2 g/kg doxycycline-impregnated food (Bio Serv catalog S3888) for 1–2 days and the GFP-β2b transgenic mice received the doxycycline-impregnated food for 1 week to maximize expression.

Generation of adenoviral vectors and infection of guinea pig ventricular cardiomyocytes. Replication-deficient adenoviral vectors expressing AID-YFP and AID-mutant YFP were generated using the AdEasy Adenoviral Vector System (Agilent Technologies) according to the manufacturer’s instructions. Briefly, sequences for AID-YFP and AID-mutant YFP were PCR-amplified and cloned into pShuttle-CMV vector. After linearization with PmeI, shuttle vectors were electroporated into BJ5183 cells containing pAdEasy-1 viral plasmid. Positive recombinants were amplified, linearized with Pac I, and transfected into AD-293 cells using the calcium phosphate precipitation method. Transfected cells were monitored for development of adenoviral plaques, after which the cells were freeze-thawed and the lysate used to infect a 10-cm dish of 90% confluent HEK293 cells. Viral expansion and purification were carried out as previously described (64).

Adult guinea pig ventricular myocytes were isolated by enzymatic digestion using a Langendorff perfusion apparatus, and cultured as previously described (27). Animal treatment and use were in accordance with a protocol approved by the Columbia University Institutional Animal Care and Use Committee. Heart cells were infected 2–3 hours after plating with 5–20 μl adenoviral vector stock (≈1011–1012 viral particles/ml).

Cellular electrophysiology. Membrane currents from isolated mouse ventricular cardiomyocytes (66) were measured by the whole-cell patch-clamp method using a MultiClamp 700B amplifier and pCLAMP 10 software (Molecular Devices) as described (65). The pipette solution contained 40 mM CsCI, 90 mM Cs gluconate, 10 mM BAPTA, 1 mM MgCl2, 4 mM Mg-ATP, 2 mM CaCl2, and 10 mM HEPES, adjusted to pH 7.2 with CsOH. After the isolated cardiomyocytes were adequately buffered with 10 mM BAPTA in the internal solution, the isolated cardiomyocytes were superfused with 140 mM TEA-Cl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, adjusted to pH 7.4 with CsOH. For experiments in tsA-201 cells, TEA-Cl was reduced to 130 mM, and 10 mM BaCl2 was used instead of CaCl2. Pipette series resistances were usually less than 1 MΩ after 60% compensation. Leak currents and capacitance transients were subtracted by a P/4 protocol. Voltages were corrected for the liquid junction potential of –10 mV. To measure Ca2+ peak currents, the cell membrane potential was held at −50 mV and stepped to +10 mV for 350 ms every 10 seconds. To evaluate the I-V relationship for Ca2+ currents, the same protocol was repeated with steps between –50 mV to +50 mV in 10-mV increments. All experiments were performed at room temperature, 22°C ± 1°C. The parameters of voltage-dependent activation were obtained using a modified Boltzmann distribution: I(V)= Gmax * (V-Erev)/[1 + exp(Vmid-V)/Vc)], where I(V) is peak current, Gmax is maximal conductance, Erev is reversal potential, Vmid is the midpoint, and Vc is the slope factor.

Fractional shortening of isolated cardiomyocytes. Freshly isolated myocytes were superfused with a Tyrode’s solution containing 1.0 mM CaCl2 and 300 nM nisoldipine. Myocytes were field stimulated at 1 Hz. Percent contraction of sarcomere length was measured using the SarcLen module (Ionoptix) and calculated as the difference of shortest sarcomere length during a contraction subtracted from the relaxed sarcomere length, divided by the relaxed sarcomere length, all averaged over at least 8 contractions.

Ex vivo cardiac contractility. The cannulated hearts were retrogradely perfused on a Langendorff system with a modified Krebs solution (118.5 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11 mM glucose, 1.8 mM Ca2+). Left ventricular (LV) pressure was measured using a balloon catheter connected to an APT-300 pressure transducer, which was connected to a PowerLab digitizer (ADInstruments). Hearts were paced at 400 beats per minute using electrodes connected to a pacing stimulator system. After initial assessment of cardiac contractility, 300 nM nisoldipine was perfused to silence endogenous Ca2+ currents. The effects of nisoldipine on contractility were assessed after at least 3 minutes and on stabilization of LV pressures. Thereafter, 200 nM isoproterenol was perfused with 300 nM nisoldipine for at least 3 minutes. Peak LV pressure during the 3-minute period was used for the assessment of β-adrenergic agonist stimulation.

Statistics. Results are mean + SEM. For multiple group comparisons, 1-way ANOVA followed by multiple comparison testing was performed. For comparisons between 2 groups, an unpaired Student’s t test was used. Statistical analyses were performed using Prism 6 (Graphpad Software). Differences were considered statistically significant at P values less than 0.05.

Data availability. The data and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure.

Study approval. The Institutional Animal Care and Use Committee at Columbia University approved all animal experiments.

Brandmayr J, et al. Deletion of the C-terminal phosphorylation sites in the cardiac β-subunit does not affect the basic β-adrenergic response of the heart and the Ca(v)1.2 channel. J Biol Chem. 2012;287(27):22584–22592.